Papillomaviruses (PV) are non-enveloped DNA viruses that induce hyperproliferative lesions of the epithelia. The papillomaviruses are widespread in nature and have been recognized in higher vertebrates. Viruses have been characterized, amongst others, from humans, cattle, rabbits, horses, and dogs. The first papillomavirus was described in 1933 as cottontail rabbit papillomavirus (CRPV). Since then, the cottontail rabbit as well as bovine papillomavirus type 1 (BPV-1) have served as experimental prototypes for studies on papillomaviruses. Most animal papillomaviruses are associated with purely epithelial proliferative lesions, and most lesions in animals are cutaneous. In the human there are more than 75 types of papillomavirus (HPV) that have been identified and they have been catalogued by site of infection: cutaneous epithelium and mucosal epithelium (oral and genital mucosa). The cutaneous-related diseases include flat warts, plantar warts, etc. The mucosal-related diseases include laryngeal papillomas and anogenital diseases comprising cervical carcinomas (Fields, 1996, Virology, 3rd ed. Lippincott—Raven Pub., Philadelphia, N.Y.).
There are more than 25 HPV types that are implicated in anogenital diseases; these are grouped into “low risk” and “high risk” types. The low risk types include HPV type 6, and type 11, which induce mostly benign lesions such as condyloma acuminata (genital warts) and low grade squamous intraepithelial lesions (SIL). In the United States, there are approximately 5 million people with genital warts of which 90% is attributed to HPV-6 and HPV-11.
The high-risk types are associated with high grade SIL and cervical cancer and include most frequently HPV types 16, 18, 31, 33, 35, 45, and 52. The progression from low-grade SIL to high-grade SIL is much more frequent for lesions that contain high risk HPV-16 and 18 as compared to those that contain low risk HPV types. In addition, only four HPV types are detected frequently in cervical cancer (types 16, 18, 31 and 45). About 500,000 new cases of invasive cancer of the cervix are diagnosed annually worldwide (Fields, 1996, supra).
Treatments for genital warts include physical removal such as cryotherapy, CO2 laser, electrosurgery, or surgical excision. Cytotoxic agents may also be used such as trichloroacetic acid (TCA), podophyllin or podofilox. Immunomodulatory agents are also available such as Interferon and imiquimod (Aldara®, 3M Pharmaceuticals). These treatments are not completely effective in eliminating all viral particles and there is either a high cost incurred or uncomfortable side effects related thereto. Also recurrent warts are common (Beutner & Ferenczy, 1997, Amer. J. Med., 102(5A):28–37).
The ineffectiveness of the current methods to treat HPV infections has demonstrated the need to identify new means to control or eliminate such infections. In recent years, efforts have been directed towards finding antiviral compounds, and especially compounds capable of interfering with viral replication (Hughes and Romanos, 1993, Nucleic Acids Res. 21:5817–5823; Clark et al., Antiviral Res., 1998, 37(2):97–106; Hajduk et al., 1997, J. Med. Chem., 49(20):3144–3150 and Cowsert et al., 1993, Antimicrob. Agents. Chemother., 37(2):171–177). To that end, it has therefore become important to study the genetics of HPVs in order to identify potential chemotherapeutic targets to contain and possibly eliminate any diseases caused by HPV infections.
The life cycle of PV is closely coupled to keratinocyte differentiation. Infection is believed to occur at a site of tissue disruption in the basal epithelium. Unlike normal cells, cellular division continues as the cell undergoes vertical differentiation. As the infected cells undergo progressive differentiation, the cellular replication machinery is maintained which allows viral DNA replication to increase, with eventual late gene expression and virion assembly in terminally differentiated keratinocytes and the release of viral particles (Fields, supra).
The coding strand for each of the papillomavirus genome contains approximately ten designated translational open reading frames (ORFs) that have been classified as either early ORFs or late ORFs. The E1 to E8 genes are expressed early in the viral replication cycle. The two late genes (L1 and L2) code for the major and minor capsid proteins respectively. The E1 and E2 gene products function in viral DNA replication, whereas E5, E6 and E7 modulate host cell proliferation. The functions of E3, E4 and E8 gene products are uncertain at present.
Studies of HPV have shown that proteins E1 and E2 are the only viral proteins required for viral DNA replication (Kuo et al., 1994, J. Biol. Chem. 30: 24058–24065). This requirement is similar to that of bovine papillomavirus type 1 (BPV-1). Indeed, there is a high degree of similarity between E1 and E2 proteins and the ori-sequences of all papillomaviruses (PV) regardless of the viral species and type (Kuo et al., 1994, supra).
When viral DNA replication proceeds in vitro, where E1 protein is present in excess, replication can proceed in the absence of E2. In vivo, in the presence of a vast amount of cellular DNA, replication requires the presence of both E1 and E2. The mechanism for initiating replication in vivo is believed to involve the cooperative binding of E1 and E2 to the origin, leading to the assembly of a ternary protein-DNA complex (Mohr et al., 1990, Science 250:1694–1699]. The E2 protein is a transcriptional activator that binds to the E1 protein and, by doing so enhances binding of E1 to the BPV origin of replication (Seo et al., 1993b, Proc. Natl. Acad. Sci., 90:2865–2869). Hence, E2 acts as a specificity factor in directing E1 to the origin of replication (Sedman and Stenlund, 1995, Embo. J. 14:6218–6228). In HPV, Lui et al. suggested that E2 stabilizes binding of E1 to the ori (1995, J. Biol. Chem. 270(45): 27283–27291 and McBride et al., 1991, J. Biol. Chem 266:18411–18414). These interactions of DNA-protein and protein-protein occur at the origin of DNA replication (Sverdrup and Myers, supra).
The ˜45 kD E2 proteins characterized from numerous human and animal serotypes share a common organization of two domains. The N-terminal transactivation domain (TAD) is about 220 amino acids and the C-terminal DNA-binding domain (DBD) is 100 amino acids in length. Both domains are joined by a flexible linker region.
E2 activates viral replication through cooperative binding with the viral initiator protein E1 to the origin of DNA replication, ultimately resulting in functional E1 hexamers. E2 is also a central regulator of viral transcription. It interacts with basal transcription factors, including TATA-binding protein, TFIIIB, and human TAFII70; proximal promoter binding protein such as Sp1; and other cellular factors such as AMF-1, which positively affect E2's transcriptional activation.
Which of these many interactions are sufficient or necessary to achieve transcriptional activation is more ambiguous. These details are consistent with the idea that enhancer binding proteins function as transcriptional activators by using specific protein-protein contacts to link components of the general transcription machinery to a promoter, with the goal of recruiting RNA polymerase II. A third function of E2 is to aid in the faithful segregation of viral DNA. The bovine papillomavirus (BPV) genome and E2 protein co-localize with host cell chromosomes during mitosis, dependent on an intact E2 TAD.
The E2 DBD dimerizes to form a β-barrel with flanking recognition helices positioned in the major grooves of the DNA binding site. In contrast, the structure of E2 TAD has remained elusive until Harris and Botchan (1999, Science, 284 (5420); 1673) provided a first model of a proteolytic fragment of HPV-18 E2 TAD by X-ray crystallography. The model suggests a cashew-shaped protein of 55 Å×40 Å×30 Å with a concave cleft on one side of the protein and ridges on the opposite surface. Harris and Botchan studied whether discrete surfaces correlated with known E2 activities and particularly identified a prominent cluster of residues constituting the inner edge of the main cavity encompassing E175, L178, Y179, and I73 defining a distinctive surface important for transcription.
Antson et al (2000, Nature, (403) 805–809) disclose the crystal structure of the complete E2 TAD from HPV-16, including a second newly identified putative E2—E2 TAD interface comprising a cluster of 7 conserved residues (R37, A69, I73, E76, L77, T81, and Q80). Anston et al suggested that Q12 and E39 may be involved in interaction with E1.
The E2 protein is considered a potential target for antiviral agents. However, drug discovery efforts directed towards E2 have been hampered by the lack of structural information of an E2 complexed with an inhibitor. Neither the model of Harris, nor that of Antson provides any information as to the localization and/or characterization of a potential inhibitor binding pocket. Structural information of the apo-E2 TAD has provided some valuable knowledge of the surface on the apo-protein but it now appears clear that this is not representative of the changes in conformation induced upon binding with an inhibitor.
The lack of specific E2 inhibitors, which is necessary for obtaining co-crystal of E2 and inhibitors, has hampered the search for the inhibitor binding pocket in E2. Thus, X-ray crystallographic analysis of such protein-inhibitor complex has not been possible.
The present invention refers to a number of documents, the contents of which are herein incorporated by reference.